The classic pathway of major histocompatibility complex class I (MHC-I) presentation is important for activation of CD8 T cells, and relies primarily on shuttling cytosolic peptides into the endoplasmic reticulum (ER) lumen by the transporter associated with antigen processing (TAP). This critical role of TAP has made it a prime target for blockade by several human viruses and downregulation in many cancers as a mechanism of evading detection by cytotoxic CD8 T cells. There are, however, other mechanisms of MHC-I presentation that bypass TAP and activate TAP-independent CD8 T cell responses. One of these is cross-presentation, where dendritic cells present peptides derived from extracellular antigens. Although TAP can be important here, cross- presentation can also take place without TAP, but the pathways involved are poorly defined. There are many disease states that result from altered TAP function and their basis is not easily explained by impairment in classic MHC-I presentation. TAP polymorphisms are associated with an increased risk for systemic lupus erythematosus (SLE), a chronic and severe systemic autoimmune disease characterized by high titers of autoantibodies against cellular constituents. TAP mutations in humans lead to TAP deficiency syndrome where surprisingly patients do not exhibit particular susceptibility to viruses but rather suffer from chronic inflammation. Finally, the successful eradication of smallpox is attributed to vaccination with cowpox virus that was recently unexpectedly shown to block TAP. Over the years, we have made significant contributions to understanding the cell biology and regulation of antigen presentation. Using genetic, cell biological, and biochemical approaches, we will pursue 3 aims seeking to delineate the pathways of TAP-independent cross-presentation: Aim 1: We have found that MHC-I reside in special compartments within dendritic cells and their trafficking from these compartments to sites of microbial antigen is regulated by Toll-like receptors, which detect microbial components. We will study how absent TAP impacts the cellular localization and trafficking of MHC-I molecules. Aim 2: We have shown that Toll-like receptors favor the cross-presentation of microbial over self-antigens. We will investigate whether cross-presentation in the absence of TAP is subject to regulation by these receptors. Aim 3: We will study the cross-presentation of both infectious viral and non-infectious cellular antigens by virally infected dendritic cells where TAP function is impaired. We would like to know whether cross- presentation can rescue MHC-I presentation of viral antigens by the very cells that are infected. We will also determine the ability of these infected cells to confine this presentation to viral and not cellular antigens. Or basic research here will enable us to identify critical points of regulation in the pathways of MHC-I presentation. We will then be able to design better vaccines for infectious diseases like malaria, tuberculosis or HIV that are best controlled by CD8 T cells. We will also be able to design strategies for intervention in autoimmune diseases like lupus where the presentation of cellular antigens is dysregulated.